Fig. 7

Inhibition of autophagy sensitizes neuroblastoma cells to chemotherapeutic agents. a SH-EP cells were grown on ibidi μ-slide 8 well™ slides and transiently transfected with the pLIB-EYFP-LC3-iresPuro plasmid. Twenty-four h after transfection the cells were treated with 20 μg/ml etoposide or with 0.25 μg/ml doxorubicin for 6 h and analyzed by live-cell fluorescence microscopy with an Axiovert200M fluorescence microscope. b SH-EP cells were treated with 20 μg/ml etoposide, 0.25 μg/ml doxorubicin, and with 100 μM CQ for 6 h. LC3-I/LC3-II expression was assessed by immunoblot analyses. GAPDH served as loading control. Densitometric analysis of LC3-II relative to GAPDH was done with the ImageJ 1.48 software. Control (Ctr.) cells were set as 100% (long exposure); CQ - treated cells were set as 100% (short exposure). Shown are mean values ± s.e.m of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05. c SH-EP cells transiently transfected with siCtr and siLC3 oligonucleotides were treated with 20 μg/ml etoposide and with 0.25 μg/ml doxorubicin for 48 h and PI-FACS analyses were performed to detect apoptotic cells. Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05, **P < 0.025. d SH-EP/shCtr, SH-EP/shDEPP (upper panel), and SH-EP/shFOXO3-17 cells (lower panel) were treated with 20 μg/ml etoposide, 0.25 μg/ml doxorubicin, and 100 μM CQ for 24 h. NB15/shCtr and NB15/shDEPP cells (upper panel) were treated with 20 μg/ml etoposide, 0.25 μg/ml doxorubicin, and 50 μM CQ for 48 h. NB15/shCtr and and NB15/shFOXO3 cells (lower panel) were treated with 10 μg/ml etoposide, 0.125 μg/ml doxorubicin, and 50 μM CQ for 48 h. PI-FACS analyses were performed to detect apoptotic cells. Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05, **P < 0.025, ***P < 0.01. e The caspase-3/7 activity assay was performed with SH-EP/shCtr, SH-EP/shDEPP, and SH-EP/shFOXO3-17 cells (left panel) treated with 20 μg/ml etoposide, 0.25 μg/ml doxorubicin, and 100 μM CQ for 24 h as well as with NB15/shCtr, NB15/shDEPP, and NB15/shFOXO3 cells (right panel) treated with 20 μg/ml etoposide, 0.25 μg/ml doxorubicin, and 50 μM CQ for 24 h. The caspase-3/7 activity was calculated between treated and untreated cells. Shown are mean values ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05, **P < 0.025